Methods for increasing the yield of fermentable sugars from plant stover

ABSTRACT

Methods for increasing yield of fermentable sugars from plant stover are provided. The methods include using plants homozygous for two brown midrib mutations, bm1 and bm3. The methods also include using plants homozygous for a mutation in a gene that results in reduced cinnamyl alcohol dehydrogenase activity, and a mutation in a gene that results in reduced 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferase activity. The methods also include using transgenic plants that have reduced cinnamyl alcohol dehydrogenase activity and reduced 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferase activity in comparison with wild-type plants.

This application claims priority to provisional patent application Ser.No. 60/676,868, filed May 2, 2005, which is incorporated by reference inits entirety.

GOVERNMENT INTERESTS

The development of the present invention was supported by DOE projectfunds. The U.S. Government may have certain rights in the inventiondescribed herein.

TECHNICAL FIELD

The present invention relates to methods for increasing the yield offermentable sugars in a plant stover.

BACKGROUND

Unlike other renewable energy sources, biomass can be converted directlyinto liquid fuels. The two most common types of biofuels are ethanol(ethyl alcohol) and biodiesel. Ethanol is an alcohol, which can beproduced by fermenting any biomass high in carbohydrates (starches,sugars, or celluloses) through a process similar to brewing beer, oncefermentable sugars have been obtained from the biomass material. Thebreakdown of the biomass into monomers (monosaccharides) requires thematerial to be softened through pretreatment, enzymes be added thathydrolyze the polymeric forms of sugars contained in the biomass intomonosaccharides, and fermentation of both the 6-carbon and 5-carbonsugars to ethanol or to other desired bio-products.

Ethanol production in the United States grew from just a few milliongallons in the mid-1970s to over 3.9 billion gallons in 2005. Nationalenergy security concerns, new Federal gasoline standards, and Governmentincentives have been the primary stimuli for this growth. Ethanol ismostly used as a fuel additive to cut down a vehicle's carbon monoxideand other smog-causing emissions. Flexible-fuel vehicles, which run onmixtures of gasoline and up to 85% ethanol, are now available.

Ethanol production received a major boost with the passage of the CleanAir Act (CAA) Amendments of 1990. Provisions of the CAA established theOxygenated Fuels Program and the Reformulated Gasoline Program in anattempt to control carbon monoxide and ground-level ozone problems. Thedesires to improve air quality and enhance energy security haveencouraged an increased demand for ethanol.

The majority of ethanol produced in the U.S. is produced from starchobtained from maize (maize) grain. It is anticipated that current starchsupplies will be insufficient to meet future demands for fermentablesugars. Ligno-cellulosic biomass, such as stover, can be used as analternative source of fermentable sugars for the production of ethanol.

Stover consists of all parts (leaves, stalks; with the exception ofkernels) of plants such as maize, sorghum, soybean, sugarcane, or otherplants that are left in the field after the harvest. This stover biomassis rich in cellulose and hemicellulose, which are cell-wallpolysaccharides that can release fermentable sugars upon treatment withenzymes (such as cellulases). The biomass conversion of maize stover iscurrently not very cost-effective.

One strategy to improve the overall efficiency of converting stoverbiomass to ethanol is to modify the chemical composition of maizestover, specifically the plant cell wall polymer lignin. Lignin has beenshown to shield the cellulose, resulting in reduced access bycellulases. In addition, lignin has been shown to be an inhibitor ofcellulases.

Brown midrib (bm) mutants of maize, easily identified by thereddish-brown color of their central leaf vein, have been known for morethan 75 years. Similar mutants have been found in sorghum, sudangrass,and pearl millet (Barriere and Argillier, 1993, Agronomie 13: 865-876).These mutants have generated significant agronomic interest becausetheir tissues are more easily digested by ruminants, providing betternutrition for livestock (Cherney et al., 1991, Adv. Agron. 46: 157-198).However, the known varieties of these plants are not widely grown asthey often suffer from slow growth, increased susceptibility to pests,and an increased tendency to lodge, all of which lead to decreasedyields.

The maize brown midrib mutations, specifically the bm1 mutation, resultin the production of abnormal lignin (Kuc and Nelson, 1964, Arch.Biochem. Biophys. 105: 103-113). Four independent maize bm mutants areknown, each affecting the lignin biosynthetic pathway. Bm1 mutants havereduced expression of cinnamyl alcohol dehydrogenase (CAD) activity(Halpin et al., 1998, Plant J. 14: 545-553). The CAD enzyme convertscinnamyl aldehydes to their alcohol derivatives in the last step ofmonolignol synthesis. The reduction of CAD activity in maize bm1 mutantsleads to large increases in the hydroxycinnamylaldehyde content of thelignin (Provan et al., 1997, J. Sci. Food Agric. 73: 133-142), and suchaldehydes have been implicated in formation of the red chromophoreresponsible, in part, for the defining coloration of the mutants(Higuchi et al., 1994, J. Biotechnol. 37: 151-158).

Two independent mutations have been identified in theO-methyltransferase (OMT) gene of bm3 maize (Vignols et al., 1995, PlantCell 7: 407-416). The bm3 gene encodes caffeic acid O-methyltransferase,or more accurately named, a 5-hydroxyconiferaldehyde/5-hydroxyconiferylalcohol O-methyltransferase (Humphreys et al., 1999, Proc Natl Acad SciUSA 96: 10045-10050). This enzyme catalyzes the methylation of the5-position hydroxyl group of 5-hydroxyconiferyl aldehyde and5-hydroxyconiferyl alcohol in monolignol synthesis. The net result ofthe bm3 mutation is a reduction in both the total amount of lignindeposited in the cell wall and a shift in lignin composition away fromsyringyl-type lignin because conversion of 5-hydroxyconiferyl aldehydeand 5-hydroxyconiferyl alcohol to sinapyl aldehyde and sinapyl alcohol,respectively is required for syringyl lignin synthesis. In bm3 mutants,the lignin also contains increased amounts of 5-hydroxyguaiacyl units,but in addition has a greatly reduced ratio of syringyl to guaiacylunits, as well as decreases in p-coumaric acid esters and overall lignincontent (Chabbert et al., 1994, J. Sci. Food. Agric. 64: 349-355).

To improve the efficiency of converting biomass to ethanol, it would beadvantageous to increase the amount of fermentable sugars in a plantstover. This invention provides that and related needs.

SUMMARY

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, and not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

A method of increasing yield of fermentable sugars in a plant stover isprovided. The method may include the steps of producing a plant stoverfrom a plant comprising a homozygous bm1 mutation and a homozygous bm3mutation, and treating plant stover cell walls with a cellulolyticenzyme to produce a fermentable sugar.

A method of increasing yield of fermentable sugars in a plant stover isprovided. The method may include the steps of producing a plant stoverfrom a plant comprising a mutation in a gene that results in a reductionin cinnamyl alcohol dehydrogenase activity and further comprising amutation in a gene that results in a reduction in O-methyltransferaseactivity, and treating plant stover cell walls with a cellulolyticenzyme to produce a fermentable sugar.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph showing glucose yields obtained from enzymaticsaccharification of unpretreated maize stover from single and double bmmutants, compared to inbred lines A619 (wild-type control).

FIG. 2 depicts: A, a graph showing the average rate of hydrolysis ofstover from wild type (♦), bm3 (▪), and bm1-bm3 (▴); B, the relativecellulose content of the stover obtained from wild type (A619), bm3, andbm1-bm3 mutant plants.

FIG. 3 depicts a graph showing xylose yields from the pretreatment ofmaize wild-type, bm2, and bm3 mutant stover.

FIG. 4 depicts graphs showing pyrolysis-gas chromatograms of stoverobtained from: A, wild-type (wt) maize inbred A619 (black) and anear-isogenic bm1 mutant (teal); B, wild-type maize inbred A619 (black)and a near-isogenic bm3 mutant (red); C, the near-isogenic bm3 (red) andbm1-bm3 (blue) mutants.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description and examples are provided to furtherillustrate the present invention and are not intended to limit theinvention beyond the limitations set forth in the appended claims.

DEFINITIONS

“Cellulolytic enzymes” include cellulose-degrading enzymes that includecellulases, cellobiohydrolases, cellobioses, and other enzymes involvedin breaking down cellulose and hemicellulose into simple sugars such asglucose and xylose. Explicitly, but not exclusively, included within theterm cellulose-degrading enzymes are those enzymes that fall under theEnzyme Classification heading EC 3.2.1.x. Preferably, “cellulolyticenzyme” means any enzyme or enzyme preparation exhibiting one or more ofthe following cellulolytic activities: endo-1,4-β-D-glucanase,exo-1,4-β-D-glucosidase, or β-D-glucosidase, which activities may bepresent to different extents in different preparations.

“Cell wall softener” means any physical, chemical, and/or biologicalmethods that render the cellulose and hemicellulose content of the cellwalls of the stover more accessible to enzymatic action. Examples ofsuch methods include treatment of the stover with water or steam,concentrated or dilute acid or alkali, milling, etc.

“Plant” means any plant species, including, but not limited to, monocotsand dicots. Examples of plants that can be used for practicing theinvention include switch grass, maize, soybean, wheat, rice, alfalfa,potato, sugar beet, barley, millet, sunflower, sorghum, canola, rice,sorghum-sudan grass, pearl millet, and sugarcane.

“Near-isogenic mutants” means a group of mutants that are geneticallyidentical except at one or a few loci. For example, near-isogenic maizebm mutants means maize lines that have a brown midrib (bm) mutation.These maize bm mutants can be single, double, triple, or more, dependingon the number of mutations in comparison to the respective brown midriballele from the wild-type maize control (A619).

“Stover” means all above-ground plant parts excluding the seed kernel.

“Fermentable sugars” means sugars that may be converted to ethanol.Glucose is an exemplary six carbon fermentable sugar derived fromcellulose. Xylose is an exemplary five carbon fermentable sugar that isderived form hemicellulose.

Overview

The present invention provides a method of increasing yield offermentable sugars in a plant stover. In one example, the methodincludes producing a plant stover from a plant comprising a homozygousbm1 mutation and a homozygous bm3 mutation. The produced plant stover isthen treated with a cellulolytic enzyme to produce a fermentable sugaror sugars. In this example, the plant stover is obtained from a maizeplant.

In another example, a method of increasing yield of fermentable sugarsin a plant stover includes producing a plant stover from a plant thatincludes a mutation in a gene that results in a reduction in cinnamylalcohol dehydrogenase activity, and also includes a mutation in a genethat results in a reduction in5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferaseactivity. The produced plant stover is then treated with a cellulolyticenzyme to produce a fermentable sugar or sugars. In this example, theplant may be maize plant.

In another example, a method of increasing yield of fermentable sugarsin a plant stover includes producing a plant stover from a plant thatincludes a down-regulated gene or gene product that results in areduction in cinnamyl alcohol dehydrogenase activity, and also includesa down-regulated gene or gene product that results in a reduction in5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferaseactivity. The produced plant stover is then treated with a cellulolyticenzyme to produce a fermentable sugar or sugars. In this example, theplant may be maize plant.

A variety of ways for down-regulation of cinnamyl alcohol dehydrogenaseand 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcoholO-methyltransferase may be used. In one example, mutations in cinnamylalcohol dehydrogenase and 5-hydroxyconiferaldehyde/5-hydroxyconiferylalcohol O-methyltransferase may be used. For example, the mutations incinnamyl alcohol dehydrogenase and5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferasemay be transcriptional, translational, post-translational, or mayinclude any combinations thereof.

Furthermore, down-regulation of cinnamyl alcohol dehydrogenase and5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol O-methyltransferasemay be induced by various genetic engineering means known in the art,for example transgenes, sense RNA, antisense RNA, RNAi, transcriptionalfactors, transgenic plants, combinations of the above, etc. Thedown-regulation may be transcriptional, translational,post-translational, or may include any combinations thereof. Thus, boththe genes and/or the respective gene products may be geneticallymodified. Similarly, down-regulation of one of the genes (e.g. cinnamylalcohol dehydrogenase) may be caused by a mutation, whereas the othergene (e.g. 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcoholO-methyltransferase) may be down-regulated by antisense RNA or in someother way. The particular mechanisms of the down-regulation of cinnamylalcohol dehydrogenase and 5-hydroxyconiferaldehyde/5-hydroxyconiferylalcohol O-methyltransferase are not important, so long as thedown-regulations result in decreased activity of both cinnamyl alcoholdehydrogenase and 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcoholO-methyltransferase, and those decreases result in an increased yield offermentable sugars as compared to wild-type plants where bm1 and bm3 arenot down-regulated.

A variety of cellulolytic enzymes also may be used. A nonlimiting listof cellulose-degrading enzymes includes those enzymes that fall underthe Enzyme Classification heading EC 3.2.1.x. Preferably, “cellulolyticenzyme” means any enzyme or enzyme preparation exhibiting one or more ofthe following cellulolytic activities: endo-1,4-β-D-glucanase,exo-1,4-β-D-glucosidase, or β-D-glucosidase, which activities may bepresent to different extents in different preparations. The activity ofcellulolytic enzymes can be assayed using methods known in the art,e.g., cellulose assays described in U.S. Pat. No. 6,818,803, hereinincorporated by reference. Some of these methods are also described inthe Examples section below.

After the production of plant stover as indicated above, and prior totreatment of the stover with a cellulolytic enzyme, the stover may betreated with a cell wall softener. This can be accomplished by any meansknown in the art, e.g. any physical, chemical, and/or biological methodsthat render the cellulose and hemicellulose content of the cell walls ofthe stover more accessible to enzymatic action. Examples of such methodsinclude treatment of the stover with water or steam, concentrated ordilute acid or alkali, milling, etc.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to limit the claimed invention.

Example 1 Production of Bm1-Bm3 Double Mutant Maize Plants

Double bm1-bm3 mutant maize plants were produced by crossing an A619inbred maize plant containing the bm3 mutation with an A619 inbred maizeplant containing the bm1 mutation. Since both these mutations arerecessive the F₂ progeny did not display a mutant phenotype because theywere heterozygous at both loci. The F₁ progeny were self-pollinated andF₂ progeny that were homozygous for both mutations were selected. The F₂progeny that were homozygous for both mutations were self-pollinated tofurther produce homozygous bm1-bm3 double mutant plants.

The bm1 mutation was first described by Eyster, 1926, Science 64: 22.Seed containing the bm1 mutation is available from the Maize GeneticsCOOP Stock Center at the University of Illinois in Urbana-Champaign,Ill. (http://w3.ag.uiuc.edu/maize-coop/mgc-info.html). The bm3 mutationwas first mentioned by Emerson et al., 1935, Maizeell Univ. Agric. Exp.Stn. Memoir 180: 1-83. Seed containing the bm3 mutation was obtainedfrom the Maize Genetics COOP Stock Center. The A619 inbred line is apublic inbred line and can be obtained through public germplasmcollections, such as the National Plant Germplasm System(http://www.ars-grin.gov/npgs/).

Example 2 Enzymatic Hydrolysis of Bm1-Bm3 Double Mutants

The bm1-bm3 double mutant maize plants were evaluated for biomassconversion properties in a lab-scale setting. The standard NationalRenewable Energy Lab procedure for enzymatic hydrolysis of stover with amix of cellulolytic enzymes was used (Brown and Torget, 1996, EnzymaticSaccharification of Lignocellulosic Biomass, LAP-009, NREL EthanolProject). The hydrolysis was performed on 300 mg of unpretreated stoverusing six filter paper units (FPU) of a commercial cellulase cocktailconsisting of a 1:1 (v/v) mix of NOVOZYME 188 and CELLUCLAST 1.5 L in 50mM citrate buffer (pH 4.8). Hydrolysis was performed at 50° C. for 24hours; the mixture was shaken during hydrolysis. The concentration ofglucose produced by this process was measured with an ACCU-CHEKADVANTAGE blood glucose meter that had been calibrated using a set ofstandard glucose solutions in the hydrolysis buffer. FIG. 1 shows theyield of glucose from the 2004 stover samples.

FIG. 1 shows glucose yields obtained from enzymatic saccharification ofunpretreated maize stover from single and double bm mutants (Summer 2004harvest), compared to inbred lines A619 (wild-type control). Thehydrolysis was performed on 300 mg stover (not pretreated), using 6 FPU(filter paper units) of a commercial cellulase cocktail consisting of a1:1 (v/v) mix of Novozyme 188 and Celluclast 1.5 L in 50 mM citratebuffer (pH 4.8). Hydrolysis was performed at 50° C. for 24 hours, whileshaking. The glucose concentration was measured with an Accu-Chek®Advantage® blood glucose meter that had been calibrated using a set ofstandard glucose solution in the hydrolysis buffer. The yield of glucoseper gram dry weight is displayed along the vertical axis.

The data in FIG. 1 show that the bm1 and bm3 mutations result in higheryields of glucose while the bm2 and bm4 mutations result in glucoseyields that are not significantly different from the wild type. Likewisethe bm2-bm3 and bm3-bm4 double mutants did not perform better than thebm3 single mutant, although compared to the bm2 and bm4 single mutants,the yield of these double mutants did benefit from the introduction ofthe bm3 mutation. The bm9-bm3 double mutant was the best performer interms of its glucose yield with on average 2.3 times as much glucose asthe wild-type control per gram dry weight under the stated assayconditions.

Combining bm1 and bm3 resulted in substantial improvement in glucoseyield. This improvement occurred without pre-treatment of the stover andresulted in glucose yields typically obtained from wild-type stoverafter pre-treatment. Thus, the combination of the bm1 and bm3 mutationscan provide significant cost savings when biomass conversion occurs on alarge scale.

Example 3 Variation in Enzyme Hydrolysis Rate

The higher yield of glucose obtained from the stover of some of themutants (FIG. 1) can be the result of (1) an increased rate ofhydrolysis, (2) an overall higher yield of glucose, or (3) thecombination of (1) and (2). To investigate this, a 168-hour hydrolysisexperiment was performed, during which hydrolysate samples werecollected at 0, 24, 48, 75 and 168 hours. This experiment was performedon wild-type, bm3 and bm1-bm3 stover (three replicate samples of eachgenotype). In addition to the hydrolysis, the cellulose content of allsamples was determined.

The results from this analysis are depicted in FIG. 2. FIG. 2A shows theaverage rate of hydrolysis of stover from wild type (A619), bm3 andbm1-bm3 mutant plants. The concentration of glucose was measured withthe Precision QID blood glucose meter which is specific for glucose.FIG. 2B shows the cellulose content of the stover obtained from wildtype (A619), bm3 and bm1-bm3 mutant plants.

Based on a t-test, the variation in cellulose content between the threedifferent genotypes was not statistically significant. For thewild-type, bm3, and bm1-bm3 stover, 20, 25 and 32%, respectively, of thecellulose was converted to glucose, expressed on the basis of the dryweight of stover (i.e. 100 mg glucose/g dry weight obtained from stovercontaining 300 mg cellulose/gram dry weight constitutes a 33%conversion). Based on these data the observed differences betweengenotypes in the rate and yield of hydrolysis between the threegenotypes are therefore best explained by scenario (3): higher rate ofhydrolysis and higher overall yield of glucose.

Example 4 Stover Pre-Treatment

The composition of the biomass as described in this invention, impactsthe severity of processing that must be carried out in the first twosteps and thereby directly impacts processing costs. Stover was selectedfrom the wild-type control and the mutants bm2 and bm3 for pretreatmentexperiments in which the experimental conditions were varied. The threemain processing parameters for acid catalyzed pretreatments were: pH,reaction temperature, and reaction time. These three factors werecombined into a single number representing the severity of thepretreatment. The combined severity factor (CSF), shown in Equation 1,fairly accurately models the improvement to digestibility frompretreatment by a single number. In batch, dilute sulfuric acidpretreatment of maize stover, Yang and Wyman (2004) have shown thatmaize stover pretreated to the same “combined severity factor” exhibitsimilar digestibility for most combinations of pH, time, and temperaturehaving a constant combined severity factor.

Equation 1:

CSF=log R _(o)−pH  (1)

-   -   where    -   R_(o)=t×e^((T-100)/14.75)    -   t=reaction time, min    -   T=reaction temperature, ° C.

For the results shown in FIG. 3, three different combined severityfactors were examined (0.03, 0.62, 1.21) which correspond to threedifferent temperatures (130° C., 150° C. and 170° C.) of 50 mM sulfuricacid for a reaction time of 5 minutes each. These severity factorsrepresent mild to moderate pretreatment conditions that are in the rangeof optimal pretreatment conditions. Maize stover samples were ground topass a 40 mesh screen and then were loaded into modified borosilicatevials and sealed. Each reactor contained a total volume of 1.5 mL. Thestover was loaded into the reactor at the equivalent of 3% by totalslurry mass.

These hydrolysis experiments were used to study the interactions betweencell wall composition and the release of monosaccharides duringpretreatment. The effect of dilute acid and liquid hot waterpretreatments is largely to solubilize the hemicellulose fraction of theplant cell wall, thus opening the structure to improved access by thecellulolytic enzymes.

FIG. 3 depicts a graph showing xylose yields from the pretreatment ofmaize wild-type, bm2, and bm3 mutant stover. Samples from threeindividual plants of each type were pretreated in duplicate. As shown inFIG. 3, the data from these experiments indicated that the mutantsbehaved similarly in the hydrolysis process to the wild-type control.The brown midrib mutations did not positively or negatively affect theacid catalyzed hydrolysis of the hemicellulose. These results indicatethat brown midrib stover performed similarly to wild type stover in acidcatalyzed pretreatments (including liquid hot water pretreatment) withrespect to the thermochemical reactions of hemicellulose hydrolysis.

Pretreatment may be carried out to minimize hydrolysis, and to “soften”the lignocellulose by pressure—cooking it in water to dissolve most ofthe hemicellulose and some of the cellulose into water solubleoligosaccharides. The lignin structure may affect this step—since achange in lignin structure could modify the covalent linkage of thehemicellulose to the lignin, with more labile bonds between the twobeing more readily broken in water at a pH maintained at pH 4 to 7 and atemperature of 140 to 200° C., thereby releasing oligosaccharides atconditions encountered in a biomass to ethanol pretreatment process. Thelignin-hemicellulose bonds will still be sufficiently stable to conferprotective effects and physical stability in the field wheretemperatures do not exceed 45° C.

Example 5 Pyrolysis-Gas Chromatograms of Stover

Further analyses of the changes in the biochemical composition of thebm1-bm3 double mutant were conducted using gas chromatography. FIG. 4shows pyrolysis-gas chromatograms of stover obtained from: A, wild-type(wt) maize inbred A619 (black) and a near-isogenic bm1 mutant (teal); B,wild-type maize inbred A619 (black) and a near-isogenic bm3 mutant(red); and C, the near-isogenic bm3 (red) and bm1-bm3 (blue) mutants.

The origin of several peaks in FIG. 4 is labeled as follows: G and Srefer to compounds in the pyrolysate derived from guaiacyl and syringylresidues in the lignin, respectively. Further, pCA and FA refer tocompounds derived from p-coumaric acid and ferulic acid, respectively.Note the similarity in the changes in composition in the bm1 and bm3stover relative to the wild-type control, specifically the reduction inthe peak heights of S- and pCA-derived compounds. The composition of thestover from the bm1-bm3 double mutant shows an even further reduction inthese compounds, as is evident from panel C. These chemical changes areconsistent with the observation that an increase in the G:S ratio in thelignin enhances the yield of fermentable sugars that is obtained fromenzymatic saccharification.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, patients, or reagents described, and as such mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is limitedonly by the claims.

We claim:
 1. A method of increasing yield of fermentable sugars in aplant stover, the method comprising: producing a plant stover from aplant comprising a homozygous bm1 mutation and a homozygous bm3mutation; and, treating the plant stover with a cellulolytic enzyme toproduce a fermentable sugar.
 2. The method of claim 1, furthercomprising treating the plant stover with a cell wall softener to softenplant stover cell walls before treating the plant stover with acellulolytic enzyme.
 3. The method of claim 1, where the plant stover isproduced by growing plants from seed and harvesting the plants.
 4. Themethod of claim 3, where the cell wall softener is selected from thegroup consisting of sulfuric acid and hot water.
 5. The method of claim1, where the plant is selected from the group consisting of switchgrass, maize, soybean, wheat, rice, alfalfa, potato, sugar beet, barley,millet, sunflower, sorghum, canola, rice, sorghum-sudan grass, pearlmillet, and sugarcane.
 6. The method of claim 5, where the plant ismaize.
 7. The method of claim 1, where the fermentable sugar is selectedfrom the group consisting of xylose and glucose.
 8. A method ofincreasing yield of fermentable sugars in a plant stover, comprising:producing a plant stover from a plant comprising a mutation in a firstgene that results in a reduction in cinnamyl alcohol dehydrogenaseactivity and further comprising a mutation in a second gene that resultsin a reduction in 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcoholO-methyltransferase activity; and treating the plant stover with acellulolytic enzyme to produce a fermentable sugar.
 9. The method ofclaim 8, further comprising the step of treating the plant stover with acell wall softener to soften plant stover cell walls before treating theplant stover cell walls with a cellulolytic enzyme.
 10. The method ofclaim 8, where the plant stover is produced by growing plants from seedand harvesting the plants.
 11. The method of claim 8, where the cellwall softener is selected from the group consisting of sulfuric acid andhot water.
 12. The method of claim 8, where the plant is selected fromthe group consisting of switch grass, maize, soybean, wheat, rice,alfalfa, potato, sugar beet, barley, millet, sunflower, sorghum, canola,rice, sorghum-sudan grass, pearl millet, and sugarcane.
 13. The methodof claim 12, where the plant is maize.
 14. The method of claim 8, wherethe fermentable sugar is selected from the group consisting of xyloseand glucose.
 15. A method of increasing the yield of fermentable sugarsin a plant stover, comprising: producing a plant stover from atransgenic plant with a reduced cinnamyl alcohol dehydrogenase activityand reduced 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcoholO-methyltransferase activity in comparison with a wild-type plant grownunder substantially similar conditions; and treating the plant stoverwith a cellulolytic enzyme to produce a fermentable sugar.
 16. Themethod of claim 15, further comprising the step of treating the plantstover with a cell wall softener to soften plant stover cell wallsbefore treating the plant stover cell walls with a cellulolytic enzyme.17. The method of claim 15, where the plant stover is produced bygrowing plants from seed and harvesting the plants.
 18. The method ofclaim 15, where the cell wall softener is selected from the groupconsisting of sulfuric acid and hot water.
 19. The method of claim 15,where the plant is selected from the group consisting of switch grass,maize, soybean, wheat, rice, alfalfa, potato, sugar beet, barley,millet, sunflower, sorghum, canola, rice, sorghum-sudan grass, pearlmillet, and sugarcane.
 20. The method of claim 19, where the plant ismaize.
 21. The method of claim 15, where the fermentable sugar isselected from the group consisting of xylose and glucose.